Limb morphogenesis requires the coordination of muscle, tendon, and cartilage development via the interactions of soluble signaling molecules and transcription factors. Our working hypothesis is that the homeodomain protein Barx2 plays multiple roles in these processes. Barx2 is expressed in muscles and tendons of the embryonic limb and is required for myogenic differentiation in vitro. Moreover, Barx2 influences and is regulated by molecules involved in limb morphogenesis. Our proposed studies are designed to understand the expression, regulation and functions of Barx2 in both muscle and tendon development. We will determine the pattern of expression of Barx2 during development of the embryonic limb with respect to specific regulators of muscle and tendon development. We will then examine whether Barx2 expression is directly regulated by muscle regulatory factors (MRFs) and tendon specification factors such as Scleraxis. Our recent studies suggest that the bone morphogenetic protein (BMP) GDF5 also regulates Barx2 expression. Because BMPs also, modulate Scleraxis expression, and GDF5 can induce ectopic tendon formation, we propose to test whether GDF5 regulates Barx2 and Scleraxis expression in limb bud cultures. Other data suggest that Barx2 influences myoblast fusion via modulation of cell adhesion and the cytoskeleton. Thus we will examine whether perturbation of Barx2 expression influences cell adhesion and cytoskeletal molecules in limb bud and myoblast cultures. We will also test whether Barx2 influences tendon development by controlling the expression of collagen I and other extracellular matrix proteins. Since developmental processes are frequently recapitulated during regeneration and repair of adult tissues, the identification of new regulatory pathways and targets for Barx2 in muscle and tendon development may lead to new avenues for therapeutic intervention in musculoskeletal disease.